The present invention relates to a prognostic method and prognostic means based on polymorphisms in the vitamin D receptor gene. In particular, the present invention relates to a method for determining susceptibility to heart disease by screening for polymorphisms in the vitamin D receptor gene.
Heart disease such as myocardial infarction is a complex phenotype caused by interaction of a number of genetic and environmental factors.1 Advances in molecular genetics have led to the identification of a number of genetic risk factors for heart disease, such as gene variants involved in thrombosis2,3 and lipid metabolism4. However, another important risk factor for heart disease such as myocardial infarction is a deregulated calcium homeostasis which is required for maintenance of neuromuscular activity5. Disturbances of the calcium balance have been implicated in hypertension, ventricular hypertrophy, aortic calcification and arrythmnias all of which are important risk factors associated with myocardial infarction.
Vitamin D is a potent regulator of bone and calcium homeostasis, as well as of cellular differentiation and replication in many tissues, and mediates its effects through the vitamin D receptor (VDR). Cloning of the vitamin D receptor has shown it to be a member of the ligand-activated superfamily, which are natural regulators of a number of physiological and developmental processes. The major physiological function of the active metabolite of vitamin D, i.e. 1,25(OH)2D3, is to maintain serum calcium levels in the normal range. This is accomplished by stimulating the small intestine to increase it""s efficiency of absorbing calcium from the diet and to mobilize calcium stores from bone. The action of the hormone is mediated by the vitamin D receptor (VDR), a steroid transcription factor that mediates hormone response by initiating gene transcription and mRNA translation6. Involvement of the vitamin D endocrine system in the etiology of heart disease was suggested by epidemiological studies which found serum levels of 25-hydroxyvitamin D3 to be reduced in myocardial infarcton patients compared with control individuals7. Furthermore, congestive hean failure associated with vitamin D deficiency has been described in a three and a half month old child8. The involvement of the vitamin D endocrine system in cardiac arrythria was suggested by case reports involving the observation of atrial flutter in a foetus with X-linked vitamin D resistant rickets9 and sick sinus syndrome that was cured by administration of vitamin D to a 77-year old caucasian woman10. Further studies in the rat have also demonstrated an important role of the vitamin D endocrine system in cardiovascular function, wherein vitamin D3-deficiency can lead to large increases in the contracile function of the heart17-20.
The above studies establish a link between the via D endocrine system and risk of heart disease in an individual. Further studies (Carling et al, JCEandM, 82 (6) 1772-1775 (1997); Carling et al, Nature Medicine, 1 (12) 13099-1310 (1995) and Carling et al, JCEandM 83 (7) 2255-2259 (1998) show an association between the b, a and T alleles of the vitamin D receptor gene and hyperparathyroidism. Hypertension is considered to be a feature of hyperparathyroidism (Boucher, B. J, of Nutr., 79 315-327 (1998). However, the genetic component of heart disease such as myocarlial infarction or cardiac arrythmia is poorly defined. As a result, methods of diagnosing risk of heart disease, or treating those suffering from heart disease, are restricted.
Thus, in a first aspect of the present invention, there is provided a method of determining susceptibility to heart disease in a subject, said method comprising analysing genetic material of a subject to determine which allele(s) of the vitamin D receptor is/are present.
The vitamin D receptor gene (12q12) comprises inherited polymorphisms between exon 7 and the 3xe2x80x2 UTR of the VDR gene. as shown in FIG. 1. These alleles are denoted B/b, A/a and T/t for restriction enzyme sites BsmI, ApaI and TaqI respectively (or enzymatic or chemical procedures with similar specificities), where a lower case letter denotes the presence of a wild type restriction site which is capable of being cleaved, and a capital letter denotes the presence of a mutant restriction enzyme site which is not capable of being cleaved by the relevant restriction enzyme. For the purposes of the present invention, determination of which alleles are present in a particular gene may be referred to as determining the genotype of a subject for a particular gene. It is apparent from the above that each copy of the vitamin D receptor gene will comprise a specific combination of the three alleles, this combination being referred to as the haplotype of the gene. For example, the haplotype may be baT, indicating the presence of cleavable BsmI and ApaI sites, and a non-cleavable TaqI site. Direct haplotyping of the VDR gene has allowed five different haplotypes to be determined, of which three are common.16 
The present invention is based on the discovery of a genetic component of heart disease. It has now been shown that presence of the b, a or T alleles, and in particular the baT haplotype of the vitamin D receptor is/are associated with increased risk of heart disease. Thus, the presence of such alleles of the vitamin D receptor gene may be used to determine susceptibility to heart disease.
Preferably, the method of the first aspect of the present invention comprises the additional step of determining whether the alleles present are associated with risk of heart disease. This may be performed by comparing the alleles present in the genetic material of the subject with genotypes of the vitamin D receptor having known degrees of risk of heart disease. For example, a visual aid detailing alleles and their relative risk of heart disease may be used to determine whether the genotype of the subject is associated with a high or low risk of heart disease.
The first aspect of the present invention may also comprise the additional step of determining aspects of calcium metabolism, such as calcium levels, in a subject. Preferably, the daily calcium intake is measured. This feature of the first aspect is based on the observation that the correlation between vitamin D receptor genotype and heart disease may be dependent upon dietary calcium intake.
The method of the first aspect may be performed in vitro. Preferably, the method is performed on tissue or fluid removed from the body of the subject. Thus, the present invention relates to a non-invasive method, the results of which provide an indication of susceptibility to heart disease but do not lead to a diagnosis upon which an immediate medical decision regarding treatment has to be made.
The present invention may be performed on any subject for whom it is desirable to determine risk of heart disease. Preferably, the subject may be a mammal. Most preferably, the method of the first aspect is performed on humans, preferably male.
Heart disease may include atrial or ventricular hypertrophy, aortic calcification, arrythmina, hypertension, and most importantly, myocardial infarction.
In a preferred feature of the first aspect of the present invention, there is provided a method of determining susceptibility to heart disease, said method comprising analysing genetic material of a subject to determine which of the B/b, A/a or T/t alleles of the vitamin D receptor gene is/are present. The method may comprise determining whether one or more of the above alleles is present. The subject may further be classified as being heterozygous or homozygous for one or more alleles. Preferably, the method comprises the additional step of determining whether the allele(s) present is/are associated with risk of heart disease, wherein presence of the b, a or T alleles is associated with increased risk of heart disease. Homozygosity for the a, b or T allele may further increase the susceptibility to heart disease in a subject.
In a preferred feature of the first aspect, there is provided a method of determining susceptibility to heart disease in a subject, said method comprising analysing the genetic material of a subject to determine the haplotype of the BsmI, ApaI and TaqI alleles at the vitamin D receptor. Preferably, said method comprises determining whether the haplotype of the subject is associated with risk of heart disease, wherein the haplotype baT is associated with high risk of heart disease. A subject homozygous for said haplotype may be at a higher risk of heart disease than those heterozygous for the haplotype.
In a preferred feature of the first aspect, there is provided a method of determining susceptibility to heart disease, said method comprising the additional step of determining the copy number of the B/b, A/a or T/t alleles or haplotype of the vitamin D receptor, where an increase in copy number is associated with increased risk of heart disease.
The present invention may be performed using any suitable method known in the art. Preferably, a tissue or fluid sample is first removed from a subject. Examples of suitable samples include blood, mouth or cheek cells, and hair samples containing roots. Other suitable samples would be known to the person skilled in the art. The genetic material is then extracted from the sample for diagnosis, using any suitable method. The genetic material may be DNA or RNA, although preferably DNA is used. For example, the DNA may be extracted using the technique described in Sambrook et al (Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratory Press). Determination of the genotype of a subject may then be carried out using the extracted DNA, employing any one of the following techniques:
Southern blot analysis following digestion with one or more appropriate restriction enzymes.
PCR amplification followed by digestion with one or more appropriate restriction enzymes and, optionally, separation of digestion products by gel electrophoresis.
Sequencing of a relevant gene fragment by any suitable method.
Visualization of heteroduplex patterns, for example on PAA or agarose gels, where different patterns may indicate the presence of one or more specific alleles.
Separation of DNA fragments using denaturing gradient gels, wherein the degree of separation will depend upon the presence or absence of one or more polymorphic restriction sites.
Separation using SSCP analysis, the patterns of which will depend upon the presence or absence of one or more polymorphic restriction sites.
Use of allele specific oligonucleotides, hybridization patterns of which will be specific for various combinations of alleles.
Methods such as OLA, Taqman or dot-blot for the detection of known mutations.
Visualization of DNA sites using fluorescent labelled probes for alleles of interest.
RFLP analysis
Where it is desirable to use particular restriction enzymes in performing the present invention, the skilled person will understand that enzymatic or chemical procedures having similar specificities may also be used. For example, restriction enzymes having similar specificity (isoschizomers) to those described herein may be used, or chemical degradation procedures with DNA or RNA cutting specificity.
Other techniques suitable for determining the genotype of a subject may be used in the present invention.
Where the haplotype of a gene is to be determined, it is preferable to use a direct haplotyping method, as described in Uitterlinden et al16. In such a method, the relevant portion of the gene is amplified and then subjected to restriction enzyme digestion, in order to determine the presence or absence of restriction enzyme sites. Thus, for example, where the haplotype of the vitamin D receptor gene is to be determined, the portion of the gene between exon 7 and the 3xe2x80x2 UTR may be amplified, and the amplified DNA digested with the BsmI, ApaI or TaqI restriction enzymes. Gel analysis may then be used to determine which alleles are present.
Preferably, a fragment may be amplified using polymerase chain reaction (PCR) techniques, to produce copies which, where the fragment is of the vitamin D receptor, are at least 1000 base pairs in length, and most preferably at least 1800 base pairs in length. PCR techniques are well known in the art, and it is within the ambit of the skilled person to identify primers for amplification of the appropriate region of the above genes, namely the region from exon 7 to the 3xe2x80x2 UTR of the vitamin D receptor gene. PCR techniques are described in EP-A-0200362 and EP-A-0201184.
In a preferred feature of the first aspect, there is provided a method of determining susceptibility to heart disease in a subject, said method comprising amplifying a fragment comprising a portion of the region from exon 7 to the 3xe2x80x2 UTR of the vitamin D receptor gene, and determining which allele(s) in the vitamin D receptor is/are present. Primers suitable for amplification of said portion of the vitamin D receptor gene would be readily available to a person skilled in the art. Examples of such primers include:
xe2x80x835xe2x80x2-CAACCAAGACTACAAGTACCGCGTCAGTGA-3xe2x80x2(SEQ ID NO:1)xe2x80x83xe2x80x831.
and/or
5xe2x80x2GCAACTCCTCATGGCTGAGGTCTC-3xe2x80x2(SEQ ID NO:2)xe2x80x83xe2x80x832.
Where the amplified portion of the gene is larger than the above defined portion of the vitamin D receptor gene containing the relevant alleles, it is preferable to avoid the inclusion of gene sequences which comprise any one of the BsmI, Apal or TaqI restriction sites.
In a second aspect of the present invention, there is provided a method of therapy, said method comprising treating a subject diagnosed as being at risk of heart disease, to reduce the risk of heart disease. Preferably, the subject is diagnosed as being at risk of heart disease in accordance with the first aspect of the present invention.
Therapy may in the form of preventative or palliative care. Suitable treatments include modifications to lifestyle, regular exercise and changes in diet. Suitable treatments, including pharmaceutical preparations, would be known to physicians and persons skilled in the art. Examples include ACE inhibitors, beta-blockers, calcium or vitamin D preparations, magnesium sulphate, thrombolytics and analgeasics.
In a third aspect of the present invention, there is provided a method of predicting the response of a subject to treatment, said method comprising analysing genetic material of a subject to determine which allele(s) of the vitamin D receptor gene is/are present. Preferably, the method includes first determining whether the subject is susceptible to heart disease. Where a subject has been determined as susceptible to heart disease, the method may further comprise administering the appropriate treatment. The present aspect of the invention is based on the observation that agents, such as calcium channel blockers, which are useful for treatment of a variety of cardiovascular diseases may also be associated with cardiovascular morbidity in some cases. The effect of an agent may therefore depend on the underlying cause of the heart disease. For example, the presence of the b, a or T alleles of the vitamin D receptor may result in modification of calcium uptake, leading to impaired cardiovascular function. Thus, in such a case it would be preferable to avoid the use of calcium channel blocking agents.
In a fourth aspect of the present invention, there is provided use of a kit to determine which allele(s) of the vitamin D receptor gene is/are present, said kit comprising (i) one or more nucleic acid primer molecules for amplification of a portion of the vitamin D receptor gene, and (ii) means for determining which allele(s) is/are present in said gene.
Preferably, the primer molecules are suitable for amplification of at least a portion of the region between exon 7 and the 3xe2x80x2UTR of the vitamin D receptor gene. Examples of suitable primers are described above.
Means for determining which allele(s) is/are present in the vitamin D receptor gene, may include any reagents or molecules necessary for use in any of the methods described above. For example, where PCR followed by DNA digestion is used, said means preferably include PCR reagents and one or more of the BsmI, ApaI, or TaqI restriction enzymes. Where the method employs Southern Blotting, heteroduplex visualization, or fluorescent labelling techniques for example, probes which bind to the appropriate regions of the vitamin D receptor gene may be included. Where necessary, such probes may be labelled to allow detection, for example by nick-translation, radio- or fluorescent-labelling, or random primer extension whereby the non-labelled nucleotides serve as a template for the synthesis of labelled molecules. Other methods of labelling probes are well known in the art.
In a preferred feature of the fourth aspect of the present invention, there is provided use of a kit further comprising means for indicating correlation between the genotype of a subject and risk of heart disease. Said means may be in the form of a chart or visual aid, which indicate that presence of the b, a or T alleles or baT haplotype of the vitamin D receptor is/are associated with heart disease such as cardiac arrythmia or myocardial infarction.
In a fifth aspect of the present invention, there is provided a kit for determining risk of heart disease in a subject, said kit comprising (i) one or more nucleic acid primer molecules for amplification of a portion of the vitamin D receptor gene; (ii) means for determining which allele(s) is/are present in said gene; and (iii) means for indicating correlation between the allele(s) and risk of heart disease.
In a preferred feature of the fifth aspect, the kit may also comprise DNA control samples, for comparison with DNA sequences of a subject. The control samples may comprise the sequence of one or more alleles of the vitamin D receptor gene, or may comprise the sequence of various haplotypes.
Preferred features of each aspect of the present invention are as for each other aspect, mutatis mutandis.